Conventional detection and identification procedures for mycobacteria require growing the organisms from patient specimens and then determining various phenotypic characteristics of the isolated organisms. These techniques may take from several days to a month or more. Molecular methods offer the hope of drastically reducing these times, potentially allowing more rapid diagnosis of mycobacterial infections. We have been working on methods to extract mycobacterial DNA directly from patient specimens (currently, we use a freeze-boil technique) and then using the polymerase chain reaction (PCR) procedure to amplify a portion of the mycobacterial 16S rRNA gene. The amplified fragment is first confirmed as being of mycobacterial origin through the use of a probe specific for the genus Mycobacterium; the particular species is identified by using species-specific probes. The procedures have already been demonstrated to work with specimens seeded with more commonly isolated species of Mycobacteria. Internal controls are used to detect PCR inhibitors in the original specimen; it is critical to detect the presence of such inhibitors because if they cannot be overcome, they render a false negative PCR result. Studies with different concentrations of the expected target may provide quantitative information about the original specimen from the amount of PCR product obtained. We are optimizing an extraction and amplification procedures to maximize the procedure's sensitivity for detecting and identifying mycobacteria in clinical specimens.